Cell crawling is central to physiological processes like wound healing, maintenance of tissue, differentiation, development (morphogenesis) and cancer metastasis. How a crawling cell positions its nucleus as it executes its normal tasks of protrusion, adhesion, and retraction of the trailing edge is poorly understood. We propose two specific aims: Aim 1: Explain how the nucleus is positioned centrally through generation of mechanical forces on the nuclear surface in crawling fibroblasts. Aim 2: Explain how the mechanical linkages between the nucleus and the F-actin cytoskeleton mediate directional persistence of fibroblast crawling. This proposal is innovative for the following reasons: 1) The majority of in vitro studies on nuclear motion have been performed in the context of motility at the edge of a wounded cell monolayer with an emphasis on initial polarization events (where the nucleus is pushed away from the leading edge). In contrast, this study will determine how an isolated crawling cell translates its nucleus in the direction of cell crawling. 2) We work from th novel conceptual view that nuclear positioning is a result of a balance of competing forward and rearward forces which can be pushing or pulling. 3) We propose to use a combination of engineering and biomolecular tools to perturb this nuclear force balance and deduce the direction and relative magnitude of the dominant cytoskeletal forces driving nuclear positioning. The medical significance of this work is due to the fact that abnormal nuclear-cytoskeletal force transfer is thought to be involved in a number of diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, Hutchinson-Gilford progeria syndrome and Dunnigan-type familial partial lipodystrophy. Although the molecular mechanism underlying these diseases remains unclear, it has been hypothesized that these diseases may result (in part) due to abnormal force transmission from the cytoskeleton to the nucleus. Our approach in this proposal relies on mutating nuclear- cytoskeletal linkers that are associated with these diseases and examining alterations in the force balance. This work therefore has both scientific and medical significance. PUBLIC HEALTH RELEVANCE: We seek to understand how cytoskeletal forces are transmitted to the nucleus, and how this force transmission mediates normal cell crawling. This knowledge will help treat the class of diseases termed as nuclear envelopathies (examples include Emery Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome) which are associated with abnormal force generation on the nucleus.